SUMMARY The central goal of this project is to examine the antiviral mechanisms in mosquitoes. As a first step, in this R21 proposal we will explore the antiviral system in the mosquito cells, Aag2. Mosquito transmission of arboviruses causes widespread and debilitating disease across the globe. Viral replication and dissemination in the mosquito are critical factors in transmission competence. RNA interference inhibits viral replication and virus-derived small RNAs accumulate during infection. Interestingly, while piRNAs are generally associated with germline defense against mobile genetic elements, in mosquitoes, virus genome-derived piRNAs (v-piRNAs) are produced during infection in somatic tissues. We discovered that Piwi4 is a viral restriction factor, and demonstrated that the piRNA pathway mediates antiviral immunity in cell culture and in mosquitoes. Furthermore, production of mature v-piRNAs requires Piwi4 and, strikingly, also depends on viral cDNA, which is synthesized by endogenous reverse transcriptases. These viral derived cDNAs are incorporated into the genome in the form of endogenous viral elements (EVEs), which are transmitted to the progeny. These results indicate that mosquitoes have evolved CRISPR-like, trans-generational adaptive antiviral immunity through the acquisition of EVEs that serve as templates for biogenesis of small antiviral RNAs. We propose to examine the role of piRNAs in mosquito antiviral immunity and to study their biological significance, mechanisms, and how ZIKV modulate piRNA immunity. Our experiments will examine the relation between ZIKV and the mosquito immune system. Specifically, we will: (1) antiviral function in the Ae. Aegypti-derived Aag2 cell and (2) identify and study ZIKV encoded piRNA suppressors.